A Mouse Mammary Tumor Virus env-Like Exogenous Sequence Is Strictly Related to Progression of Human Sporadic Breast Carcinoma

Abstract

A viral etiology of human breast cancer (HBC) has been postulated for decades since the identification of mouse mammary tumor virus (MMTV). The detection of MMTV env-like exogenous sequences (MMTVels) in 30% to 40% of invasive HBCs increased attention to this hypothesis. Looking for MMTVels during cancer progression may contribute to a better understanding of their role in HBC. Herein, we analyzed HBC preinvasive lesions for the presence of MMTVels. Samples were obtained by laser microdissection of FFPE tissues: 20 usual-type ductal hyperplasias, 22 atypical ductal hyperplasias (ADHs), 49 ductal carcinomas in situ (DCISs), 20 infiltrating ductal carcinomas (IDCs), and 26 normal epithelial cells collateral to a DCIS or an IDC. Controls included reductive mammoplastic tissue, thyroid and colon carcinoma, and blood samples from healthy donors. MMTVels were detected by fluorescence-nested PCR. DNA samples from the tissues of nine patients were analyzed by real-time quantitative PCR, revealing a different viral load correlated with stage of progression. Furthermore, as never previously described, the presence of MMTVels was investigated by chromogenic in situ hybridization. MMTVels were found in 19% of normal epithelial cells collateral to a DCIS or an IDC, 27% of ADHs, 82% of DCISs, and 35% of IDCs. No MMTVels were found in the control samples. Quantitative PCR and chromogenic in situ hybridization confirmed these results. These data could contribute to our understanding of the role of MMTVels in HBC. A viral etiology of human breast cancer (HBC) has been postulated for decades since the identification of mouse mammary tumor virus (MMTV). The detection of MMTV env-like exogenous sequences (MMTVels) in 30% to 40% of invasive HBCs increased attention to this hypothesis. Looking for MMTVels during cancer progression may contribute to a better understanding of their role in HBC. Herein, we analyzed HBC preinvasive lesions for the presence of MMTVels. Samples were obtained by laser microdissection of FFPE tissues: 20 usual-type ductal hyperplasias, 22 atypical ductal hyperplasias (ADHs), 49 ductal carcinomas in situ (DCISs), 20 infiltrating ductal carcinomas (IDCs), and 26 normal epithelial cells collateral to a DCIS or an IDC. Controls included reductive mammoplastic tissue, thyroid and colon carcinoma, and blood samples from healthy donors. MMTVels were detected by fluorescence-nested PCR. DNA samples from the tissues of nine patients were analyzed by real-time quantitative PCR, revealing a different viral load correlated with stage of progression. Furthermore, as never previously described, the presence of MMTVels was investigated by chromogenic in situ hybridization. MMTVels were found in 19% of normal epithelial cells collateral to a DCIS or an IDC, 27% of ADHs, 82% of DCISs, and 35% of IDCs. No MMTVels were found in the control samples. Quantitative PCR and chromogenic in situ hybridization confirmed these results. These data could contribute to our understanding of the role of MMTVels in HBC. See related Commentary on page 1588 See related Commentary on page 1588 Despite the fact that breast carcinoma represents the most frequent cancer in women and that it has been largely studied worldwide for many decades, its etiology is largely unknown. The hereditary transmission of pathogenetic mutations of some predisposing genes, such as BRCA1 and BRCA2, is the only factor recognized as causative in this disease.1Petruccelli N. Daly M.B. Feldman G.L. Hereditary breast and ovarian cancer due to mutations in BRCA1 and BRCA2.Genet Med. 2010; 12: 245-259Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar Although the pathogenetic role of estrogens is well demonstrated, sound evidence of their etiological effect is still lacking.2Russo J. Russo I.H. The role of estrogens in the initiation of breast cancer.J Steroid Biochem Mol Biol. 2006; 102: 89-96Crossref PubMed Scopus (435) Google Scholar A minimal percentage of cases are caused by radiation.3Ronckers C.M. Erdmann C.A. Land C.E. Radiation and breast cancer: a review of current evidence.Breast Cancer Res. 2005; 7: 21-32Crossref PubMed Scopus (256) Google Scholar On the other hand, in mice, the etiological role of mouse mammary tumor virus (MMTV) in the development of tumors of the mammary gland has been demonstrated for a long time.4Bittner J.J. Some possible effects of nursing on the mammary gland tumor incidence in mice.Science. 1936; 84: 162Crossref PubMed Scopus (378) Google Scholar Notably, much of what is known about the pathogenesis of human breast carcinoma (HBC) was learned by the experimental model of the MMTV-induced mouse mammary tumors.5Ross S.R. MMTV infectious cycle and the contribution of virus-encoded proteins to transformation of mammary tissue.J Mammary Gland Biol Neoplasia. 2008; 13: 299-307Crossref PubMed Scopus (31) Google Scholar, 6Cardiff R.D. Kenney N. Mouse mammary tumor biology: a short history.Adv Cancer Res. 2007; 98: 53-116Crossref PubMed Scopus (49) Google Scholar In particular, the concept of cancer progression and the recognition of the so-called preinvasive lesions as morphological steps of its development are based on the MMTV model.7Medina D. Premalignant and malignant mammary lesions induced by MMTV and chemical carcinogens.J Mammary Gland Biol Neoplasia. 2008; 13: 271-277Crossref PubMed Scopus (26) Google Scholar Moreover, the promotional role of estrogens was built on the observations conducted in mice.8Medina D. De Ome K.B. Effects of various oncogenic agents on tumor-producing capabilities of series D BALB-c mammary nodule outgrowth lines.J Natl Cancer Inst. 1970; 45: 353-363PubMed Google Scholar, 9Fernandez S.V. Russo J. Estrogen and xenoestrogen in breast cancer.Toxicol Pathol. 2010; 38: 110-122Crossref PubMed Scopus (164) Google Scholar The strong similarities between the human and the murine disease led to a half-century quest for a possible viral etiology of breast carcinoma in women. MMTV viral antigens were found in human breast tumors,10Mesa-Tejada R. Keydar I. Ramanarayanan M. Ohno T. Fenoglio C. Spiegelman S. Immunohistochemical detection of a cross-reacting virus antigen in mouse mammary tumors and human breast carcinomas.J Histochem Cytochem. 1978; 26: 532-541Crossref PubMed Scopus (32) Google Scholar MMTV particles were described in human cells and milk,11Moore D.H. Sarkar N.H. Kelly C.E. Pillsbury N. Charney J. Type B particles in human milk.Tex Rep Biol Med. 1969; 27: 1027-1039PubMed Google Scholar, 12Al-Sumidaie A.M. Leinster S.J. Hart C.A. Green C.D. McCarthy K. Particles with properties of retroviruses in monocytes from patients with breast cancer.Lancet. 1988; 1: 5-9Abstract PubMed Scopus (36) Google Scholar and MMTV sequences were found in humans.13Callahan R. Drohan W. Tronick S. Schlom J. Detection and cloning of human DNA sequences related to the mouse mammary tumor virus genome.Proc Natl Acad Sci U S A. 1982; 79: 5503-5507Crossref PubMed Scopus (91) Google Scholar Unfortunately, these data were never considered conclusive mainly because of their scarce reproducibility. Moreover, until a few years ago, the involvement of viruses in the etiology of human solid tumors did not receive much attention and the researchers were not stimulated in this direction. Finally, the identification of endogenous MMTV sequences in humans was a discouraging element. In 1995, Wang et al14Wang Y. Holland J.F. Bleiweiss I.J. Melana S. Liu X. Pelisson I. Cantarella A. Stellrecht K. Mani S. Pogo B.G. Detection of mammary tumor virus env gene-like sequences in human breast cancer.Cancer Res. 1995; 55: 5173-5179PubMed Google Scholar selected a 660-bp region of MMTV with a nucleotide sequence identity of only 16% to human endogenous retroviral element K10, which is highly similar to MMTV, and 90% to 98% identical to the MMTV envelope (env) gene. By using MMTV-specific primers located in the 660-bp region, Wang et al14Wang Y. Holland J.F. Bleiweiss I.J. Melana S. Liu X. Pelisson I. Cantarella A. Stellrecht K. Mani S. Pogo B.G. Detection of mammary tumor virus env gene-like sequences in human breast cancer.Cancer Res. 1995; 55: 5173-5179PubMed Google Scholar identified an MMTV env gene–like exogenous sequence (MMTVels) in 38% of a series of human infiltrating breast carcinomas. On the other hand, MMTVels was found in only 2% of normal human breast samples. Several other groups15Etkind P. Du J. Khan A. Pillitteri J. Wiernik P.H. Mouse mammary tumor virus-like ENV gene sequences in human breast tumors and in a lymphoma of a breast cancer patient.Clin Cancer Res. 2000; 6: 1273-1278PubMed Google Scholar, 16Ford C.E. Faedo M. Crouch R. Lawson J.S. Rawlinson W.D. Progression from normal breast pathology to breast cancer is associated with increasing prevalence of mouse mammary tumor virus-like sequences in men and women.Cancer Res. 2004; 64: 4755-4759Crossref PubMed Scopus (50) Google Scholar, 17Lawson J.S. Tran C.E. Rawlinson W.D. W. of mouse mammary virus gene sequences may associated with of human breast cancer.J Pathol. 2006; PubMed Scopus (26) Google Scholar were to these were A. R. M. The mouse mammary tumor virus-like env gene sequence is not in breast cancer of Google Scholar, A. S. R. J. for DNA of exogenous mouse mammary tumor virus in human breast cancer 2007; PubMed Scopus Google Scholar These could the of in and the that are only a few of env sequences and were not highly In et M. A. R. C. sequences in human breast cancer: a microdissection Pathol. 2006; PubMed Scopus Google Scholar from this a to these based on the of a laser microdissection and a highly fluorescence-nested PCR. The MMTVels was detected in of a series of normal breast tissues and other of human tumors were for the that a virus could in the etiology of HBC is much attention that in the In most I. M. A. J. Holland J.F. Pogo B.G. of viral particles from of human breast cancer Res. 2007; PubMed Scopus Google Scholar, I. J. A. Holland J.F. Pogo B.G. Detection of human mammary tumor virus proteins in human breast cancer 2010; PubMed Scopus Google Scholar has been to the of viral proteins in tumor the of MMTV to human cells was S. Mouse mammary tumor virus human Res. 2005; PubMed Scopus Google Scholar MMTV, in breast human could as a or as an to some steps of cancer such as or The of MMTV exogenous sequences during cancer progression could their role in HBC. the presence of MMTVels in a series of preinvasive breast lesions ductal epithelial atypical ductal epithelial ductal carcinoma in situ and infiltrating ductal carcinoma Moreover, the presence of the exogenous viral sequence in normal preinvasive and infiltrating from the was an of MMTV env-like sequences in HBC progression. quantitative PCR the PCR the to our we the presence of the MMTV env-like viral sequence by chromogenic in situ hybridization which detected viral sequence hybridization in the of DCIS and and were and to the of and of cells and were obtained by laser DNA was from and MMTV env-like sequences were detected by fluorescence-nested PCR and M. A. R. C. sequences in human breast cancer: a microdissection Pathol. 2006; PubMed Scopus Google Scholar In nine real-time was The collateral is to normal epithelial cells in the a preinvasive or an infiltrating carcinoma, and preinvasive lesions in the an infiltrating a ductal epithelial by and of the has and of DCIS in or or in an not DCIS of or cells with and with solid and with cells with and with a or and of a between and W.D. of human and of breast PubMed Scopus Google Scholar, A of the of for atypical and of the PubMed Scopus Google Scholar, The and of breast ductal carcinoma in situ and atypical ductal and Cancer Res. PubMed Scopus Google Scholar The included 26 collateral to DCIS or 20 to DCIS or 22 to DCIS or 49 DCISs, 20 of which were collateral to and 20 IDCs. the DCISs, were were and was well The control included 20 from reductive mammoplastic thyroid colon and DNA from blood samples from healthy blood donors. The DNA of a mouse by MMTV was the A laser was to the epithelial to were from using a for a of to and cells were of the long of the with this was in of M. A. R. C. sequences in human breast cancer: a microdissection Pathol. 2006; PubMed Scopus Google Scholar samples were in K. Samples were for PCR the DNA samples were in with the samples. The and of DNA were by and DNA was for the of PCR by as The cycle were as cycle for for for and for and a for in PCR of and and U DNA samples were considered of PCR were on a PCR was to the presence of the MMTV env-like were on an DNA The of were on the of the sequence in were to our M. A. R. C. sequences in human breast cancer: a microdissection Pathol. 2006; PubMed Scopus Google Scholar to it to mainly of the of the to a of The primers a from nucleotide to of and the primers a and of the primers for the PCR were as and the PCR, the sequence was the as the previously the sequence was were in PCR and U was DNA in the PCR and of PCR in the The was as cycle for and for for and for and a for PCR and DNA samples were included samples in were analyzed by and as in an The was from a in PCR and in of PCR from were with of and of for samples were an and analyzed using MMTVels PCR and MMTV were on using the and on an sequence was by using the to MMTV sequences in DNA from nine patients was analyzed by In and were in the other only DCIS and were a PCR was using and of MMTV for the env region PCR, The viral load was as the of The of DNA from which is a was as an to of human DNA and of MMTV env were included as on MMTV env-like and PCR were in the to experimental control DNA from a mouse known to with the virus and a control were included in PCR The were on the The were by the the known of or and MMTV DNA was by the The MMTV was by of a PCR using primers previously Wang Y. Melana Pelisson I. Holland J.F. Pogo B.G. of a in human breast cancer.Cancer Res. Google Scholar The PCR was conducted in a U of DNA of PCR of of of and Wang Y. Melana Pelisson I. Holland J.F. Pogo B.G. of a in human breast cancer.Cancer Res. Google Scholar and of mouse DNA for the MMTV The PCR were as for by of for and for The was using the The were as were and to were and was by using a The were in to the in a for the was on the for The and hybridization were by of MMTV on the for and for using a Finally, the were detected by for which a using of and control were The control were from a known to for MMTVels in the PCR The control were from the with hybridization of the MMTV from breast for the presence of viral and from colon cancer A of was to the in viral between the and of tumor progression in the of laser microdissection that only cells were selected for DCIS laser microdissection with DCIS laser microdissection The presence and the of MMTVels in normal breast and in of breast cancer progression are described and in and in exogenous MMTVels were found in 19% of collateral to DCIS or IDC. The 20 cases of normal breast samples obtained by reductive were ductal epithelial the 20 cases of were for exogenous MMTV env-like sequences were found in 27% of samples of breast carcinoma, MMTVels were found in 82% of of DCIS 35% of of samples were for the cases of infiltrating thyroid carcinoma, the cases of colon and the DNA samples of healthy were different obtained from the normal breast and were from patients with DCIS for MMTVels. In samples were in cases to DCIS and were the normal were in cases to normal and DCIS were and was and in cases to only were In the patients to with for in normal were the normal and were cases of in which were the was in cases and in cases to and 26 to In nine cases of in which only normal gland were to of the were and was In the cases with only to were and to were and of MMTVels in 49 and in and were as of DCISs, of of of of and DCISs, of not in a were as of DCISs, of of of of and DCISs, of not the were to MMTV, the were and to the region of the MMTV sequence and MMTV and nucleotide to MMTV and human mammary tumor virus env Finally, were found the sequences were with the human sequences in that these were not of human or endogenous was conducted on DNA from DCIS and from different was as the gene represents the DNA The of the DNA for DCIS and of samples is was a in MMTV between the was conducted on DNA from and from different patients In the PCR was in and in IDCs. In was an in MMTV DNA the progression from normal to DCIS a of the virus in the invasive in sequences were detected by in DCIS samples. of the cases The were the the of The of from with an of in are much cells with hybridization in the in the DCIS cases not The etiology of the breast carcinoma is with the of C.M. Erdmann C.A. Land C.E. Radiation and breast cancer: a review of current evidence.Breast Cancer Res. 2005; 7: 21-32Crossref PubMed Scopus (256) Google Scholar The role of estrogens has been well for many sound evidence of their possible causative is still lacking.2Russo J. Russo I.H. The role of estrogens in the initiation of breast cancer.J Steroid Biochem Mol Biol. 2006; 102: 89-96Crossref PubMed Scopus (435) Google Scholar The to breast cancer pathogenesis that the between ductal and carcinoma is to a based on C.M. T. M.B. M. C.A. A. C. D. of human breast 2000; PubMed Scopus Google Scholar, M. W. of breast Cancer PubMed Scopus Google Scholar, M. C. S. 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W. J. Lawson J. Rawlinson W. Mouse mammary tumor virus sequences in breast from healthy Cancer PubMed Scopus Google Scholar the presence of sequences in from healthy women. Unfortunately, of these or is to that the most cancer in women is caused by On the other hand, is element this the and of data to this the MMTV env-like it possible that some were the of the of env sequences and A M. A. R. C. sequences in human breast cancer: a microdissection Pathol. 2006; PubMed Scopus Google Scholar from this that a these particular, a laser microdissection to a and of tumor cells and a highly fluorescence-nested PCR for DNA On the of of the data in the it is to that the presence of exogenous sequences of MMTV in infiltrating HBC is and that the percentage of cases is between 30% and A C. the for human retroviruses in Mol Biol 2008; PubMed Scopus Google Scholar on human viruses that our microdissection M. A. R. C. sequences in human breast cancer: a microdissection Pathol. 2006; PubMed Scopus Google Scholar with in situ PCR C.E. Faedo M. Rawlinson W.D. Mouse mammary tumor virus-like and DNA are found in cells of human breast Cancer Res. 2004; PubMed Scopus Google Scholar the of DNA and with tumor cells and the that sequences are in some MMTVels is in human breast it could an or In other it could as a an etiological a pathogenetic The of exogenous viral sequences during the of the disease an contribution to the for the to our the presence of MMTVels in preinvasive infiltrating and normal MMTV exogenous viral sequences are in are in 27% of ADHs, 82% of DCISs, and 35% of IDCs. collateral to DCIS is in 19% of in it is obtained by reductive in healthy women. These data well in with the model of progression for breast cancer: normal and J.S. S.R. The and of breast in Cancer Res. PubMed Scopus Google Scholar, J.S. and of invasive breast and their evidence to the concept of breast Pathol. 2008; PubMed Scopus Google Scholar, J.F. on atypical epithelial and ductal carcinoma in Full Text PDF PubMed Scopus Google Scholar, C.M. M. K.B. between DCIS and invasive Cancer PubMed Scopus Google Scholar, Y. S. S. C.M. P. A. D. carcinoma in situ and the of during breast cancer Res. 2008; Scholar, S. M. of in ductal as a breast carcinoma 2008; Google Scholar and DCIS are considered of and DCIS are included in the of ductal epithelial represents a in human as the of a In it is considered to not related to as by its the of and the of in and are a few cases that are with the of of in to of with few similar to in mainly in to an invasive carcinoma, as by the of in breast and other In the few epithelial cells by the in a breast the of to in a few which is a frequent The of the with the of to ADHs, in which is MMTVels are in normal to in a percentage of cases and not been found in normal of normal breast obtained by reductive or in not associated with IDCs. MMTVels is in ADHs, in and in to a similar to that of The presence of MMTVels in and in normal and that these exogenous sequences an to steps of The 35% in is with The of in normal breast not associated with the few cases in normal to and the of in are in with the model and the of The only is the of MMTV env-like sequences in DCIS with a in the lesions and and IDC. the of and the in this the of the results. A possible for the strong in cases from DCIS to that MMTVels are for for of to IDCs. Moreover, MMTVels could as a of the of IDCs. A of tumor cells MMTV antigens as a of their identification and by the These data are by obtained by that the MMTV env-like sequence is and that is a from DCIS to IDC. In the from normal cells to The in the of cases from normal cells to was previously demonstrated by et C.E. Faedo M. Crouch R. Lawson J.S. Rawlinson W.D. Progression from normal breast pathology to breast cancer is associated with increasing prevalence of mouse mammary tumor virus-like sequences in men and women.Cancer Res. 2004; 64: 4755-4759Crossref PubMed Scopus (50) Google Scholar during the of from to On the other the lesions studied and are not with the preinvasive lesions analyzed Furthermore, and most in this we for the to our the presence of the MMTV viral sequence by in which we a of from DNA of a mouse with the MMTV as in the presence of the viral hybridization in the of DCIS and The from cells and the of found by Liu et Wang Y. Melana Pelisson I. Holland J.F. Pogo B.G. of a in human breast cancer.Cancer Res. Google Scholar in conducted in situ hybridization on breast cancer in In the of cells that viral hybridization is much in the cases in the DCISs, the concept of a of viral sequence load with the progression of the disease the that the viral sequences are only in the transformation Finally, a not of this is the that MMTV env gene-like sequences detected in In exogenous MMTV sequences are in the steps of breast cancer their possible role in the transformation as an etiological On the other hand, the strong of cases from DCIS to that are not for the progression of the data to to is a viral etiology of HBC. The of the MMTV virus from patients and the detection of its Mouse Mammary in or of Commentary the by et this on the detection of sequences in human breast cancer PDF